A fluorescently labeled, biologically active derivative of thyrotropin-releasing hormone (TRH) will be used to localize TRH receptors on pituitary tumor cells, to study ligand-induced receptor aggregation, and to investigate possible internalization of the TRH-receptor complex. Localization of TRH receptors will also be carried out by the unlabeled antibody method using a TRH-dinitrophenol derivative and antiserum to dinitrophenol, and a two-headed TRH derivative and antiserum to TRH. The importance of receptor clustering in early TRH actions, stimulation of prolactin release and uridine uptake, will be tested. The concentration of TRH receptors is regulated by thyroid hormones and other drugs. To study the mechanism by which these agents control receptor levels, density labeling techniques will be applied to measure the rate of TZH receptor synthesis and degradation under various conditions. A histidyl-azide-TRH derivative will be tested as a photoaffinity label for TRH receptors. Centrifugal elutriation will be used to separate pituitary tumor cells in different phases of the cell cycle and the separated populations will be tested for hormone synthesis and TRH receptors and responsiveness. The fraction of normal pituitary cells with TRH receptors will be determined by fluorescence microscopy and the fluorescent TRH derivative will be incubated with pituitary cells from animals subjected to various hormonal manipulations. The fluorescence activated cell sorter will be used to separate normal pituitary cells which bind fluorescent TRH derivatives. Populations of cells with and without TRH receptors will be strained for cell type by the unlabeled antibody technique and tested for responsiveness to TRH. The effects of physiological concentrations of thyroid hormones on TRH receptors and responses, thyrotropin and prolactin secretion will be investigated in normal pituitary cell cultures.